Target C5a in Inflammation
C5a is a 74 amino acid spanning split product of its “mother molecule” C5 and represents one endpoint of the complement activation cascade. It can be generated through activation of at least three well-described pathways (the alternative, the classical and the MBL pathway). All pathways merge at the level of C3, forming the C5− or alternative C5 convertase leading to cleavage of C5 into C5a and C5b. The latter binds with C6, C7, C8 and multiple C9 molecules ultimately leading to formation of pores in e.g. bacterial membranes (terminal Membrane Attack Complex=MAC). C5a is generated when the complement system is activated in settings of inflammation and other immunological and inflammatory disorders/diseases.
Among the complement activation products, C5a is one of the most potent inflammatory peptides, with a broad spectrum of functions (Guo and Ward, 2005). C5a exerts its effects through the high-affinity C5a receptors (C5aR and C5L2) (Ward, 2009). C5aR belongs to the rhodopsin family of G-protein-coupled receptors with seven transmembrane segments; C5L2 has a similar structure but appears not to be G-protein-coupled. It is currently believed that C5a exerts its biological functions primarily through C5a-C5aR interaction, as few biological responses have been found for C5a-C5L2 interaction. However, latest reports demonstrate signaling also through C5L2 activation (Rittirsch and others, 2008).
C5aR is widely expressed on myeloid cells including neutrophils, eosinophils, basophils, and monocytes, and non-myeloid cells in many organs, especially in the lung and liver, indicative of the importance of C5a/C5aR signaling. Widespread up-regulation of C5aR expression occurs during the onset of sepsis, and blockade of C5a/C5aR interaction by anti-C5a, or anti-C5aR antibodies, or C5aR antagonists renders highly protective effects in rodent models of sepsis (Czermak and others, 1999; Huber-Lang and others, 2001; Riedemann and others, 2002).
C5a has a variety of biological functions (Guo and Ward, 2005). C5a is a strong chemoattractant for neutrophils and also has chemotactic activity for monocytes and macrophages. C5a causes an oxidative burst (O2 consumption) in neutrophils and enhances phagocytosis and release of granular enzymes. C5a has also been found to be a vasodilator. C5a has been shown to be involved in modulation of cytokine expression from various cell types and to enhance expression of adhesion molecule expression on neutrophils. High doses of C5a can lead to nonspecific chemotactic “desensitization” of neutrophils, thereby causing broad dysfunction. Many inflammatory diseases are attributable to the effects of C5a, including sepsis, acute lung injury, inflammatory bowel disease, rheumatoid arthritis and others. In the experimental setting of sepsis, exposure of neutrophils to C5a can lead to neutrophil dysfunction and paralysis of signaling pathways, leading to defective assembly of NADPH oxidase, paralysis of MAPK signaling cascades, a great depression of oxidative burst, phagocytosis and chemotaxis (Guo and others, 2006; Huber-Lang and others, 2002). Thymocytes apoptosis and delayed neutrophil apoptosis are two important pathogenic events for sepsis development, which are dependent on the presence of C5a. During experimental sepsis, C5a up-regulates β2-integrin expression on neutrophils to promote cell migration into organs, one of the major causes for multi-organ failure (MOF). It is also found that C5a is attributable to the activation of the coagulation pathway that occurs in experimental sepsis. C5a stimulates the synthesis and release from human leukocytes of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8, and macrophage migration inhibitory factor (MIF). Given that complement activation is an event occurring during the onset of acute inflammation, C5a may come into play before emergence of most of the inflammatory “cytokine storm”. It appears that C5a plays a key role in orchestrating and amplifying the performance of the cytokine network and the formation of systemic inflammatory response syndrome (SIRS).
In the immunological regulatory network tailing to the adaptive immunity, C5a affects the crosstalk between dendritic cells (DC) and δεT cells, and this may result in a large production of inflammatory mediators such as IL-17 (Xu and others, 2010). An essential role for C5a has been established and defined in the generation of pathogenic Th17 responses in systemic lupus erythematosus (SLE) (Pawaria and others, 2014). In addition, it has been reported that C5a is a key regulator for Treg cells offering a powerful suppressive effect for Treg propagation and induction (Strainic and others, 2013). Given the fact that Treg and TH17 are the essential players in the autoimmune disease setting, inhibition of C5a signaling would be expected to significantly reduce overactive immune status in the autoimmune diseases.
IFX-1
IFX-1 is a chimeric monoclonal IgG4 antibody which specifically binds to the soluble human complement split product C5a. IFX-1 is composed of 1328 amino acids and has an approximate molecular weight of 148,472 Daltons. The CDR and FR sequences of IFX-1 are disclosed in Table 3 below.
IFX-1 is expressed in a mammalian CHO cell line as recombinant protein and finally formulated in a phosphate buffered saline solution for intravenous administration. The binding of this antibody to human C5a facilitates a highly effective blockade of C5a-induced biological effects by disabling C5a binding to and reacting with its corresponding cell-bound receptors.
Various nonclinical studies were conducted to assess pharmacological and toxicological aspects of IFX-1, which can be divided into in vitro/ex vivo tests and in vivo studies including GLP toxicology studies in cynomolgus monkey (using IFX-1). None of the conducted nonclinical tests and studies revealed any toxicological or safety concerns for IFX-1. Human Phase I trial indicated that safety laboratory parameters, vital signs and ECG parameters showed no clinically relevant time or dose-related changes.
In vitro analysis of IFX-1 demonstrates a strong binding capacity to soluble human C5a as well as a high blocking activity of C5a-induced biological effects such as lysozyme release from human neutrophils or CD11b up-regulation in neutrophils in human whole blood. One IFX-1 antibody reaches the capability of neutralizing the effects of 2 molecules C5a with close to 100% efficiency in experimental in vitro settings. Clinical trials with IFX-1 have been ongoing to test its clinical efficacy in several inflammatory diseases including septic organ dysfunction and complex cardiac surgery.
Neutrophils
Neutrophils, terminally differentiated cells with a short lifespan in circulation, are the most abundant leukocytes in the human body. As a first line of defense against invading microorganisms, neutrophils are characterized by their ability to act as phagocytic cells, release lytic enzymes from their granules and produce reactive oxygen species upon stimulation. In addition to microbial products, other stimuli such as immune complex can also induce the respiratory burst in neutrophils, leading to enhanced inflammation and the recruitment of inflammatory cells (Kaplan, 2013).
After infiltrating into inflamed tissues, neutrophils engage in many other cell types, such as macrophages, dendritic cells (DCs), natural killer cells, lymphocytes and mesenchymal stem cells, regulate innate and adaptive immune responses. For instance, neutrophils can modulate DC maturation and the proliferation and polarization of T cells, and they can also directly prime antigen-specific T-helper type 1 and T-helper type 17 cells (Abi Abdallah and others, 2011). A variety of stimuli induce neutrophil degranulation, including C5a, formyl-methionyl-leucyl-phenylalanine (FMLP), lipopolysaccharide, platelet activating factor, and Tumor necrosis factor (TNF) (Kaplan, 2013). The contents released from degranulation and oxidative species together with cytokines and chemokines resulted from neutrophil activation are the primary inflammatory mediators that cause tissue damage, and this mechanism is believed to be attributable to many types of inflammatory tissue injury.
Hidradenitis Suppurativa (HS)
HS is a chronic devastating skin disorder affecting areas rich in apocrine glands, and it is considered as one of neutrophil-associated cutaneous inflammatory diseases. Nodules appear in the affected areas, and they progressively become swollen and rupture with the release of pus. This process occurs repeatedly leading to sinus tract formation and scars (Jemec, 2004). This disease course creates a frustrating situation for the patients but also for physicians. The point prevalence is reported to range between 1% and 4% (Jemec and others, 1996).
The exact pathophysiology of HS is not well defined. Smoking, dietary habits and genetic predisposition have all been linked with HS (Kurzen and others, 2008; Slade and others, 2003). The percentage of NK cells was increased and that of CD4-lymphocytes decreased compared to healthy controls probably implying the existence of an autoimmune predilection for the disorder. IL-1β and IL-17 have been found to be upregulated in the lesion of HS, being associated with the activation of inflammasome (Lima and others, 2016). Hidradenitis suppurativa (HS) is presented with the high number of neutrophil infiltrates in the inflamed skin, especially in the late stage of disease (Lima and others, 2016; Marzano, 2016). Activated neutrophils could be an important effector cell type causing tissue damage through direct harmful effect or indirect regulatory effect toward other effect cells such as active T cells and TH17 in this disease setting.
A hypothesis for the implication of some autoimmune or autoinflammatory mechanism in the pathogenesis of HS has been created over the last years. The hypothesis is further reinforced by positive results from the administration of TNF antagonists in prospective, placebo-controlled studies, which result in the approval of Adalimumab (an antibody directed against tumor necrosis factor α) in patients with moderate to severe HS. One major, yet unanswered question is how neutrophils are recruited to the affected skin lesion and to what extent activated neutrophils would contribute to the disease development.
The wide range of possible pathogenic mechanisms suggested by different studies may imply that HS is associated with host mechanisms rather than exogenous factors. Taking into account of the paradox that both anti-infectious (antibiotics) and pro-infectious (anti-TNF, corticosteroids, immunosuppressive drugs) therapies may be helpful, HS may appear as an auto-inflammatory disease based on a defect in the hair follicle innate immunity (Revuz, 2009), which is supported by the fact that pro-inflammatory cytokines such as interleukin (IL)-1β, and tumor necrosis factor-α (TNF-α) are markedly increased in lesional and perilesional skin (Wollina and others, 2013).
Neutrophilic Dermatoses
The neutrophilic dermatoses (ND) are a group of disorders characterized by skin lesions for which histologic examination reveals intense inflammatory infiltrates composed primarily of neutrophils with no evidence of infection. ND mainly include Sweet syndrome (SS), pyoderma gangrenosum (PG), subcorneal pustular dermatosis (SPD), other well-defined entities, and their atypical or transitional forms (Prat and others, 2014). Hidradenitis suppurativa (HS) has recently been assigned to the family of ND based on the high number of neutrophil infiltrates observed in the inflamed skin (Lima and others, 2016; Marzano, 2016).
Pyoderma gangrenosum (PG) and hidradenitis suppurativa (HS) are prototypic neutrophilic dermatoses that are regarded as autoinflammatory disease in origin with the hallmark of the accumulation of neutrophils in the skin (Braun-Falco and others, 2012; Marzano and others, 2014). Autoinflammatory Syndrome represents an emerging group of inflammatory conditions that are distinct from autoimmune, allergic, and infectious disorders. From a pathophysiological perspective, all the autoinflammatory syndromes such as PAPA (pyogenic arthritis, PG and acne), PASH (PG, acne and hidradenitis suppurativa) or PAPASH (pyogenic arthritis, acne, PG and hidradenitis suppurativa) share common mechanisms consisting of over-activation of the innate immune system and ‘sterile’ neutrophil-rich cutaneous inflammation (Cugno and others, 2017).
Neutrophils and Autoimmune Diseases
Autoimmune diseases are defined by defective differentiation of self and non-self molecules, leading to inappropriate recognition of self molecules and tissues as foreign structures, and concomitant immune attack against host organs. The pathogenesis of autoimmune diseases can generally be divided into two phases, immunization phase and effector phase. Immunization phase is characterized by the emergence of autoreactive T-lymphocytes. Those T-cells then trigger a secondary response leading to tissue damaging phase by activating various other cell types (B-cells, cytotoxic T-cells, NK-cells, neutrophils, macrophages, osteoclasts, fibroblasts, etc.). The activation of those effector cells by the autoreactive T cells can be considered as the effector phase which can be mediated by multiple levels including autoantibody production, cytokine networks or direct cell-cell contacts (Nemeth and Mocsai, 2012).
The role of neutrophils in the pathophysiological development of autoimmune diseases has been limitedly defined, but increasingly appreciated. Neutrophils could participate in the multiple steps of the autoimmune disease process, including antigen presentation, regulation of the activity of other immune cell types, and direct tissue damage. Neutrophils can expose/release autoantigens when activated, or when dying by apoptosis, or during formation of neutrophil extracellular traps (NETs). They can also contribute to tissue deposition of autoantibodies or, as an effector cell type, they can induce tissue damage themselves. Accumulative studies have demonstrated that neutrophils play an active role in the development of autoimmune diseases, such as, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), bullous pemphigoid, epidermolysis bullosa acquisita, ANCA-associated vasculitis, familial Mediterranean fever, cryopyrin-associated periodic disorders (CAPS) and gout, etc. (Nemeth and Mocsai, 2012; Nemeth and others, 2016). As the skin being an easy target for immune responses, cutaneous inflammation is one of most frequent syndromes presented by these autoimmune diseases.